Methods for screening HIF like ouabain-resistant Na+-K+-ATPase agents

ABSTRACT

The present invention relates to rapid, quantitative, specific, high through-put methods for screening test substances for their ability to inhibit activity of an ouabain-resistant Na + -K + -ATPase involved in a variety of biological processes such as regulation of osmotic balance and cell volume, maintenance of the resting membrane potential, establishment of the ionic composition of cerebrospinal fluid and aqueous humor, electrical activity of muscle and nerve, and receptor-mediated endocytosis, cardiac muscle contractility, neurotransmitter metabolism and vascular muscle cell contraction. These methods can be employed to identify compounds for use in therapeutic applications for disease processes in which dysfunction of the Na + -K + -ATPase contributes to a pathological process. The present invention also includes kits which are used in the methods provided herein. The present invention further includes methods of treating or preventing diseases or disorders which are associated with dysfunction of the Na + -K + -ATPase.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant, R01HL52282, from the National Heart, Lung and Blood Institute of theNational Institutes of Health. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Na⁺-K⁺-ATPASE is a plasma membrane-associated enzyme which is encoded bya multigene family. Activity of the Na⁺-K⁺-ATPase provides gradients ofNa⁺ and K⁺ that are essential for maintaining cellular homeostasis.(Levenson, Rev. Physiol. Biochem. Pharmacol. 123:1-45, 1994). The iongradients established by the Na⁺-K⁺-ATPase play a central role inregulating osmotic balance, cell volume, and maintaining the restingmembrane potential. Na⁺-coupled transport of nutrients, establishment ofthe ionic composition of cerebrospinal fluid and aqueous humor,electrical activity of muscle and nerve, and receptor-mediatedendocytosis are all processes which depend on the activity of theenzyme.

Compounds have been identified that inhibit Na⁺-K⁺-ATPase, and at leastsome of these compounds, plant-derived cardiac glycosides like ouabainand digoxin, have found therapeutic utility, such as treatment ofcongestive heart failure. However, ouabain can also have deleteriousside effects that have not been associated with the use of an endogenouscompound, hypothalamic inhibitory factor (HIF), which also has beenfound to have an inhibitory effect on Na⁺-K⁺-ATPase.

Sensitivity of the Na⁺-K⁺-ATPase to ouabain and HIF lies within the asubunit of the enzyme. To date, all α2 and α3 isoforms and α1 isoformsfrom most mammals and other organisms tested are sensitive to ouabain,i.e., can be inhibited at relatively low ouabain concentrations.However, certain α1 isoforms are resistant to ouabain.

A need exists to identify other inhibitors of Na⁺-K⁺-ATPase which, likeHIF, will have fewer potential side-effects than ouabain, and relatedcardiac glycosides obtained from plants.

SUMMARY OF THE INVENTION

The present invention is directed to methods and kits for screening atest substance for HIF inhibitory activity of an ouabain-resistantNa⁺-K⁺-ATPase and for treating or preventing diseases or disorders withsubstances identified by the screening methods described herein.

In one embodiment, the method includes contacting an ouabain-resistantNa⁺-K⁺-ATPase with a test substance under conditions suitable formeasuring or detecting ouabain-resistant Na⁺-K⁺-ATPase activity. Theouabain-resistant Na⁺-K⁺-ATPase inhibitory activity of the testsubstance is measured or detected. The level of activity of theouabain-resistant Na⁺-K⁺-ATPase measured or detected in the presence ofthe test substance is then compared with the ouabain-resistantNa⁺-K⁺-ATPase activity measured or detected in the presence of HIF undercomparable conditions, thereby determining whether the test substanceexhibits HIF inhibitory activity.

In another embodiment, the method includes contacting theouabain-resistant Na⁺-K⁺-ATPase with a test substance in the presence ofATP and a non-ATP substrate, under conditions suitable for measuring ordetecting ouabain-resistant Na⁺-K⁺-ATPase activity. Oxidation of thenon-ATP substrate is measured or detected to determine the ATPaseactivity. This activity is compared to the activity measured or detectedusing again the oxidation of a non-ATP substrate contacted withouabain-resistant Na⁺-K⁺-ATPase and ATP in the presence of HIF undercomparable conditions, thereby determining whether the test substanceexhibits HIF inhibitory activity.

In still another embodiment, the method includes contactingouabain-resistant Na⁺-K⁺-ATPase with a test substance in the presence ofATP, wherein the terminal phosphate (P₃) is labeled, under conditionssuitable for measuring or detecting liberated labeled P₃. Labeled P₃liberated from the ATP substrate is measured or detected. The measuredor detected liberated labeled P₃ is compared with measured or detectedlabeled P₃ liberated by contact of ouabain-resistant Na⁺-K⁺-ATPase withHIF in the presence of ATP, wherein the terminal phosphate is labeled,and under comparable conditions, thereby determining whether the testsubstance exhibits HIF activity.

In a further embodiment, a liposome containing, or a cell containing orexpressing, ouabain-resistant Na⁺-K⁺-ATPase is contacted with a testsubstance in the presence of a compound comprising Rb⁺, under conditionssuitable for isotopic Rb⁺ uptake by the liposomes or the cells. Theamount of isotopic Rb⁺ present in the liposomes or cells is comparedwith isotopic Rb⁺ measured or detected in liposomes or cells obtained bycontacting liposomes containing, or cells containing or expressing,ouabain-resistant Na⁺-K⁺-ATPase with HIF in the presence of a compoundcomprising isotopic Rb⁺, under comparable conditions, therebydetermining whether the test substance exhibits HIF inhibitory activity.

A kit of the invention includes an isolated ouabain-resistantNa⁺-K⁺-ATPase, or reconstituted liposomes or cells containing orexpressing, an ouabain-resistant Na⁺-K⁺-ATPase. The kit also includesHIF, a non-ATP substrate, ATP or labeled ATP, and a compound containingisotopic Rb⁺.

In another embodiment, the kit includes an isolated ouabain-resistantNa⁺-K⁺-ATPase, ATP, NADH and HIF.

In a further embodiment, the kit includes an isolated ouabain-resistantNa⁺-K⁺-ATPase, ATP in which the P₃ is labeled and HIF.

In yet another embodiment, the kit includes reconstituted liposomescontaining, or cells containing or expressing, ouabain-resistantNa⁺-K⁺-ATPase, a compound containing isotopic Rb⁺ and HIF.

A method of the invention treats or prevents a disease or disorder byadministering, to a subject to whom such treatment or prevention is inneed thereof, an effective therapeutic amount of a biologically activesubstance identified by any of the methods described herein forscreening a test substance as exhibiting HIF inhibitory activity.

The present invention encompasses rapid, quantitative, specific, highthrough-put methods for screening test substances such as drugs, ligands(natural or synthetic), ligand antagonists, peptides, small organicmolecules and the like, for their ability to inhibit activity of anouabain-resistant Na⁺-K⁺-ATPase involved in a variety of biologicalprocesses. Examples of such biological processes include the regulationof osmotic balance and cell volume, maintenance of the resting membranepotential, establishment of the ionic composition of cerebrospinal fluidand aqueous humor, electrical activity of muscle and nerve, andreceptor-mediated endocytosis, cardiac muscle contractility,neurotransmitter metabolism and vascular muscle cell contraction.Identification of other substances that, like HIF, inhibitouabain-resistant Na⁺-K⁺-ATPase, may lead to treatment or prevention ofdisorders or diseases without the side effects characteristic ofouabain.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention will now be moreparticularly described and pointed out below and in the claims. It willbe understood that the particular embodiments of the invention are shownby way of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

The present invention is directed to methods and kits for screening atest substance for HIF inhibitory activity of an ouabain-resistantNa⁺-K⁺-ATPase and for treating or preventing diseases or disorders withsubstances identified by the screening methods described herein. It isbelieved that other compounds which, like HIF, inhibit ouabain-resistantNa⁺-K⁺-ATPase may also share a low toxicity profile.

Sensitivity of Na⁺-K⁺-ATPase to ouabain and hypothalamic inhibitoryfactor (HIF) lies within the a subunit of the enzyme. However, certainα₁ isoforms isolated from rats, mice, species of amphibians such as thetoad Bufo marinus, and species of butterflies such as the Monarchbutterfly Danaus plexippus, are resistant to ouabain (Levenson R., Rev.Physiol. Biochem. Pharmacol., 123:1-45, 1994; Holzinger et al., FEBS314:477-480, 1992; and Jaisser et al., J. Biol. Chem. 267:16895-16903,1992). In order to inhibit these ouabain-resistant Na⁺-K⁺-ATPases, a 100fold or higher concentration of ouabain than that capable of inhibitingouabain sensitive Na⁺-K⁺-ATPase generally is needed. In addition, anumber of mutations have been found to be able to convert anouabain-sensitive Na⁺-K⁺-ATPase into an ouabain-resistant one (Lingrelet al., The Sodium Pump (Bamberg and Schoner, Ed.) pp. 276-286, 1994).Most of these mutations are located within the extracellular domains orat the borders of the extracellular domains. While inactivation of theouabain-resistant α₁ isoform (obtained from rat kidney) requiresconcentrations of ouabain 10²-10³ greater than that for inactivating theouabain-sensitive α₂/α₃ isoforms (obtained from rat brain axolemma)(Sweadner, Biochem. Biophys. Acta. 988:185-220, 1989), such is not thecase for hypothalamic inhibitory factor (hereinafter HIF). Ferrandi andco-workers isolated HIF from bovine and rat hypothalamus (Farrandi etal., Am. J. Physiol. 263:F739-F748, 1992), and discovered chat HIF couldinhibit ouabain-resistant Na⁺-K⁺-ATPase at physiological dosages.

In one embodiment of the present invention, an ouabain-resistantNa⁺-K⁺-ATPase is contacted with a test substance under suitableconditions. An example of a suitable medium is an aqueous medium forwhich the ouabain-resistant Na⁺-K⁺-ATPase is active (Sweadner, K., J.Biol. Chem., 260:11508-11513, 1985).

Ouabain-resistant Na⁺-K⁺-ATPase proteins and nucleic acids can beobtained by methods known in the art. The ouabain-resistantNa⁺-K⁺-ATPase can be derived, isolated or obtained from various targetcell types. Examples of target cell types include, but are not limitedto, kidney, heart, pineal gland, skeletal muscle, retina horizontal,retina Muller cells, brain cortical astrocytes, cerebellar granuleneurons, cortical neurons and Hippocampal neurons. In a specificembodiment, the target cells are derived, isolated or obtained from apatient exhibiting a disease state that is related to the dysfunction ofthe ouabain-resistant Na⁺-K⁺-ATPase (Decollogne et al., The Sodium Pump,Bamberg and Schoner (eds) pp. 812-815, 1994). Examples of the diseasestare include, but are not limited to, cardiac malfunctions such ascongestive heart failure, paroxysmal atrial tachycardia or atrialfibrillation, edematous disorders such as congestive heart failure,cirrhosis of the liver and nephrotic syndrome, and hypotension. In aspecific embodiment, the ouabain-resistant Na⁺-K⁺-ATPase is derived froma rodent, an amphibian or a butterfly species. For example, a suitableouabain-resistant Na⁺-K⁺-ATPase can be derived from a mouse, a rat, atoad Bufo marinus or a Monarch butterfly Danaus plexippus.Alternatively, the ouabain-resistant Na⁺-K⁺-ATPase can be obtained byconverting or mutating an ouabain-sensitive Na⁺-K⁺-ATPase into anouabain-resistant one. The ouabain-resistant Na⁺-K⁺-ATPase can beobtained by changing or substituting one or both border amino acidresidues of the H1-H2 extracellular domain of a subunit of anouabain-sensitive Na⁺-K⁺-ATPase to a charged amino acid residue,including a positively or negatively charged amino acid. Additionalexamples of mutations that confer ouabain resistance to an otherwiseouabain-sensitive Na⁺-K⁺-ATPase is disclosed in Lingrel et al., TheSodium Pump (Bamberg and Schoner, Ed.) pp. 276-286, 1994, the content ofwhich is incorporated herein by reference in its entirety. Thenucleotide sequence for rat, butterfly Monarch Danaus plexippus and toadBufo marinus α1 Na⁺-K⁺-ATPase is disclosed in Shull et al., Biochemistry25:8125-8132, 1986; Jaisser et al., J. Biol. Chem. 267:16895-16903, 1992and Holzinger et al., FEBS 314:477-480, 1992, respectively, theteachings of which are incorporated in their entirety herein byreference.

Cells of animal origin, particularly, rodent species such as mouse orrat, amphibians such as toad Bufo marinus and butterfly species such asMonarch butterfly Danaus plexippus can serve as a nucleic acid sourcefor the isolation of a isoforms of Na⁺-K⁺-ATPase nucleic acids. The DNAcan be obtained by standard procedures known in the art from cloned DNA(e.g., a DNA “library”), chemical synthesis, cDNA cloning, or by thecloning of genomic DNA, or fragments thereof, purified from the desiredcell (see, for example, Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A PracticalApproach, MRL Press, Ltd., Oxford, U.K. Vol. I, II; the entire teachingsof which are incorporated in its entirety by reference herein). Clonesderived from genomic DNA can contain regulatory and intron DNA regionsin addition to coding regions; clones derived from cDNA or RNA containexon sequences. Whatever the source, the gene is generally molecularlycloned into a suitable vector for propagation of the gene.

In the molecular cloning of the gene from cDNA, cDNA can be generatedfrom totally cellular RNA or mRNA by methods that are known in the art.The gene can also be obtained from genomic DNA, where DNA fragments aregenerated (e.g. using restriction enzymes or by mechanical shearing),some of which will encode the desired gene. The linear DNA fragments canthen be separated according to size by standard techniques, includingbut not limited to, agarose and polyacrylamide gel electrophoresis andcolumn chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing all or a portion of the Na⁺-K⁺-ATPase gene can beaccomplished in a number of ways known to those skilled in the art(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover,D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,Oxford, U.K. Vol. I, II; Shilo and Weinberg, 1981, Proc. Natl. Acad.Sci. USA 78:6789-6792; the entire teachings of which are incorporated byreference herein in its entirety).

Ouabain-resistant Na⁺-K⁺-ATPase proteins and derivatives, analogs andfragments thereof can be obtained by methods known in the art, includingbut not limited to, recombinant expression methods, purification fromnatural sources, and chemical synthesis (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985,DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I,II; Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792;the entire teachings of which are incorporated by reference herein inits entirety).

The test substance that is contacted with the ouabain-resistantNa⁺-K⁺-ATPase by the method of the invention can be a chemical compound,such as organic molecules, inorganic molecules, organic/inorganicmolecules, proteins, peptides, polysaccharides, saccharides,glycoproteins, nucleic acid strands or oligonucleotides likedeoxyribonucleic acid (hereinafter “DNA”) and ribonucleic acid(hereinafter “RNA), or mixture of compounds like in the case of alibrary of test substances, a natural extract or tissue culture extract,whose effect on an ouabain-resistant Na⁺-K⁺-ATPase is determined by oneor more of the methods described herein. The test substance can be anorganic molecule, such as a steroid, cardiac glycoside or structurallysimilar compound, or a combinatorial library of such compounds. Ouabainitself is not considered a “test substance” as that term is used herein.

Activity of an ouabain-resistant Na⁺-K⁺-ATPase is measured or detectedby methods known in the art, which methods include, but are not limitedto, coupled-enzyme assays (Haupert, G. et al,. Am. J. Physiol.,247:F919-F924, 1984), ATP hydrolysis assays (Doucet et al., Am. J.Physiol., 237(2):F105-F113, 1979), and ion transport assays (Cantielloet al., Am. J. Physiol. , 255:F574-F580, 1988). The assay can bequantitative or qualitative.

The level of activity measured or detected for ouabain-resistantNa⁺-K⁺-ATPase, when contacted with the test substance, is compared tothe ATPase activity in the Ad presence of HIF under comparableconditions.

HIF is isolated from mammalian organs, tissues or cell types containingHIF. In a specific embodiment, the HIF is isolated from hypothalamus(e.g., bovine hypothalamus) according to the procedures disclosed inU.S. Pat. No. 5,716,937 or Tymiak et al., Proc. Natl. Acad. Sci. USA90:8189-8193, 1993; the teachings of which are incorporated herein byreference in their entirety.

HIF generally is attributed with having the followingcharacteristics: 1) it is a regio-chemical or stereo-chemical isomer ofouabain; 2) it specifically and reversibly binds ouabain-resistantNa⁺-K⁺-ATPase; and 3) after naphtholylation, it yields a CD spectrumsubstantially different from that of ouabain, see U.S. Pat. No.5,716,937. It should be recognized that an HIF analogue, derivative,solvate or related compound having HIF inhibitory activity on anouabain-resistant Na⁺-K⁺-ATPase can also be used, although it is idealthat HIF itself is used.

“Comparable conditions” according to the method of the invention aredefined as the conditions of an assay conducted in substantially thesame manner as a referent assay. That is to say, if an assay is to bethe object of comparison (i.e., if a comparison of activity of the testsubstance is to be made to the activity of a reference compound such asHIF), then the assay of activity of the reference material would be thereferent assay under conditions equivalent to, or the same as,conditions under which the activity of the test substance was assayed.The concentration(s) of the test substance used in methods describedherein generally are equivalent or the same as that of the HIF. Incomparing the activity of the test substance with that of HIF, adetermination is made as to whether the test substance inhibits theactivity of the same ouabain-resistant Na⁺-K⁺-ATPase (e.g., rat kidney)to the same extent as HIF inhibitory activity.

“HIF inhibitory activity” is defined herein to mean the inhibition ofthe ouabain-resistant Na⁺-K⁺-ATPase exhibited by HIF. For example, atest substance can be determined to have HIF inhibitory activity if itinhibits the same ouabain-resistant Na⁺-K⁺-ATPase (e.g., rat kidney) tothe equivalent or same degree as HIF at the equivalent or sameconcentration and under equivalent or the same conditions under whichHIF exhibits inhibitory activity. In one embodiment, a positive resultis found when the level of ouabain-resistant Na⁺-K⁺-ATPase activitymeasured when the ouabain-resistant Na⁺-K⁺-ATPase is contacted with thetest substance is the same as or less than that measured when theouabain-resistant Na⁺-K⁺-ATPase is contacted with HIF under comparableconditions. A test substance that would be of particular interest can beone that exhibits, for example, from about the same to about ten timesthe HIF inhibitory activity.

In comparing the activity of an ouabain-resistant Na⁺-K⁺-ATPase (or“pump”) measured when the pump is contacted with a test substance withthat when the pump is contacted with HIF, it is unnecessary to measurethe activity of the ouabain-resistant Na⁺-K⁺-ATPase in parallel,although such parallel measurement is considered to be within the scopeof the invention.

In another embodiment, ouabain-resistant Na⁺-K⁺-ATPase inhibitoryactivity can be measured or detected using a coupled-assay, wherein theouabain-resistant Na⁺-K⁺-ATPase is contacted with a test substance inthe presence of ATP and a non-ATP substrate. More specifically, a testsubstance is preincubated in a buffer containing ouabain-resistantNa⁺-K⁺-ATPase and ATP. The preincubation is carried out in the presenceof chelators such as norepinephrine, BSA, and EGTA to avoidinterferences by ionic species present in the enzyme or HIF fractions,such as vanadate, free fatty acids, and bivalent cations. Afterpreincubation, the incubating solution is added to a prewarmed readingsolution containing buffering agents (physiological pH is used), ATP,and a non-ATP substrate, such as NADH. The enzymatic reaction isconducted at a temperature where the enzyme is active, e.g. from about25° C. to about 40° C. for a period of time where activity, if present,is detected for about 1 second to about 5 minutes or more. A moredetailed description of this assay can be found in Haupert, G., et al.,Am. J. Physiol., 247:F919-F924, 1984; the teachings of which areincorporated by reference in their entirety.

In one embodiment, the non-ATP substrate is NADH which is oxidized toform NAD⁺. The oxidation of the non-ATP substrate and, thus, enzymeactivity, is measured either by an increase of the amount (oraccumulation) of the oxidized substrate, or a decrease of the amount (ordepletion) of the non-ATP substrate. The oxidation reaction is monitoredconstantly, intermittently or finally by techniques known in the art.For example, spectrophotometry can detect a decrease of the amount ofthe non-ATP substrate or an increase of the amount of the oxidizednon-ATP substrate. The enzymatic activity can be calculated from theslope of the oxidation reaction. Enzymatic activity typically isexpressed as μmol (of the decrease of the non-ATP substrate or theincrease of the oxidized form of the non-ATP substrate)/μg protein/min.In one particular embodiment, the inhibitory activity of the testsubstance is expressed as percent of the control sample assay, where thecontrol sample assay is performed under the same conditions but in theabsence of either the test substance or HIF. In one embodiment, theconversion of NADH to NAD⁺ is measured by a decrease of the amount ofthe NADH. In another embodiment, the decrease of the amount of the NADHis measured by a decrease in absorbance at 340 nm. An example of asuitable assay employing NADH can be found in Haupert, G., et al., Am.J. Physiol., 247:F919-F924, 1984; the teachings of which areincorporated in their entirety.

A comparison is then made between the measurement, or detection, ofoxidation of the non-ATP substrate when the ouabain-resistantNa⁺-K⁺-ATPase is in the presence of the test substance with themeasurement, or detection, of oxidation of the non-ATP substrate whenthe ouabain-resistant Na⁺-K⁺-ATPase is in the presence of HIF, therebydetermining whether the test substance exhibits HIF inhibitory activity.

In another embodiment, the ouabain-resistant Na⁺-K⁺-ATPase is contactedwith a test substance in the presence of ATP, wherein the terminalphosphate group (P₃) is labeled. The P₃ of ATP is labeled in such a waythat the label does not substantially interfere with the hydrolysis ofATP by the ouabain-resistant Na⁺-K⁺-ATPase. The label of the P₃ isselected from, for example, the group consisting of a radiolabel, afluorescent label, a luminescent label and an enzymatic label. In oneembodiment, the labeled ATP is [³²P]ATP.

In this embodiment, ouabain-resistant Na⁺-K⁺-ATPase activity can bemeasured by an ATP hydrolysis assay. Typically, a suitable ATPhydrolysis assay includes preincubating a test substance in aphysiological buffer containing isolated ouabain-resistantNa⁺-K⁺-ATPase. Parallel experiments can be carried out in the presenceof different buffer solutions or chelators during preincubation, such asnorepinephrine, EDTA, EGTA, glutathione, mercaptoethanol, and BSA, toevaluate the ionic requirements for the binding of the test substance,and to determine if, in comparison to HIF, chelators interfere with theinhibitory activity of the test substance. After the preincubationperiod, the incubation solution containing ATP (in which P₃ is labeled)is allowed to proceed for a sufficient time to cleave labeled P₃ from[³²P]ATP at a temperature, for example, in a range from about 25° C. toabout 37° C., and from about 2 to 30 minutes. The reaction is thenterminated by, for example, the addition of perchloric acid, or someother suitable method.

The liberated, labeled P₃ is then separated from the rest of thereaction mixture by a suitable separation technique, such as is known inthe art. In one embodiment, the liberated labeled P₃ is separated fromthe rest of the reaction mixture by centrifugation. In a specificembodiment, the liberated labeled P₃ is separated from the rest of thereaction mixture by centrifugation in the presence of activatedcharcoal.

The amount of the liberated labeled P₃ is measured. The amount of theliberated ³²P (or the amount of [³²P]ATP remaining) can be measured by,for example, liquid scintillation counting. The inhibitory activity ofthe test substance and HIF is expressed as percent of the controlsample; that is, the assay can be performed in the absence of either thetest substance or HIF.

A comparison is then made between the ouabain-resistant Na⁺-K⁺-ATPaseactivity in presence of the test substance with that of the ATPaseactivity incubating in the presence of HIF under comparable conditions.The comparison of activity between the two conditions is through themeasurement or detection of liberated labeled P₃.

In another embodiment of the present invention, isotopic Rb⁺ is used inscreening a test substance for HIF inhibitory activity. A screeningmethod is used to examine the putative inhibitory effect of a testsubstance upon the ouabain-resistant Na⁺-K⁺-ATPase by analyzing theuptake of the isotopic Rb⁺ into, for example, reconstituted liposomescontaining, or cells expressing or containing, the ouabain-resistantNa⁺-K⁺-ATPase. Examples of ⁸⁶Rb⁺-containing compounds include, but arenot limited to, Rb⁺ salts, such as RbCl, RbBr and RbAc. In a specificembodiment, the isotopic Rb⁺ is ⁸⁶Rb⁺. An example of a suitable assayemploying isotopic Rb⁺ is described in Anner et al., Am. J. Physiol.,258:F144-F153, 1990; the teachings of which are incorporated byreference in their entirety.

Purified ouabain-resistant Na⁺-K⁺-ATPase can be reconstituted intophosphatidylcholine liposomes using methods in the art, such as thecholate-dialysis method disclosed by Anner and Moosmayer in Biophys.Res. Commun. 129:102-108, 1985. Cells containing ouabain-resistantNa⁺-K⁺-ATPase, such as rat kidney cells, can be prepared by standardtechniques known in the art (see, for example, Shyjan and Levenson,Biochemistry 28:4531-4535, 1989; the entire teachings of which areincorporated by reference). To measure ion transport activity of thereconstituted liposomes containing, or cells containing or expressing,ouabain-resistant Na⁺-K⁺-ATPase, freshly reconstituted liposomes orisolated cells are washed with a physiological solution and resuspended.A test substance is preincubated with the washed reconstituted liposomesor cells. After preincubation, a compound containing an isotopic Rb,such as ⁸⁶RbCl, is added. The incubation is stopped after sufficienttime, form about 1 to 5 minutes at about 25° C. to about 37° C., toallow internal accumulation of ⁸⁶Rb⁺. The liposomes or cells areseparated from the medium and the amount of isotopic Rb⁺ present in theliposomes or cells are counted. The inhibitory activity of the testsubstance and HIF is expressed as percent of the control sample, carriedout in the absence of the test substance and HIF.

Rb⁺ present in liposomes containing, or in the cells expressing orcontaining, the ouabain-resistant Na⁺-K⁺-ATPase is separated from theunincorporated or liberated compound containing Rb⁺ by a suitableseparation technique, such as is known in the art. In one embodiment,the Rb⁺ present in the cells is separated from the unincorporated orliberated compound containing Rb⁺ by centrifugation through, forexample, an oil layer, such as a phthalate oil layer. In anotherembodiment, the Rb⁺ present in the liposomes is separated from theunincorporated or liberated compound containing Rb⁺ by chromatography.In one embodiment, the amount of the ⁸⁶Rb⁺ present in the liposomes orthe cells is measured by a gamma counter.

A comparison is then made between the ouabain-resistant Na⁺-K⁺-ATPaseactivity incubated in presence of the test substance with that of theATPase activity incubated in the presence of HIF under comparableconditions. The comparison of activity between the enzyme beingincubated with the test substance versus HIF is made through themeasurement or detection of ⁸⁶Rb⁺ present in the liposomes or cellsexpressing or containing ouabain-resistant Na⁺-K⁺-ATPase.

Inhibitors of an ouabain-resistant Na⁺-K⁺-ATPase identified by thescreening methods disclosed above can be employed to treat or preventcertain diseases or disorders, such as a cardiac malfunction, to producea positive inotropic effect. Examples of disorders or diseases that canbe treated by test substances identified by the method of the inventionare taught in U.S. Pat. No. 5,716,937, the entire content of which isincorporated herein by reference. U.S. Pat. No. 5,716,937 disclosesmethods of treating cardiac malfunction such as congestive heartfailure, paroxysmal atrial tachycardia and atrial fibrillation,edematous disorders such as congestive heart failure, cirrhosis of theliver or nephrotic syndrome and hypotension, by administering a positiveinotropic effect-producing glycosidic HIF. The inhibitors identified bythe invention can be isolated and/or prepared and incorporated intosuitable pharmaceutical compositions by suitable methods, such as thoseknown in the art, for oral, parenteral (or intravenous) delivery.

The present invention also includes kits which can be employed using themethods disclosed herein. In specific embodiments, kits of the inventioncan include all or some of the following items: (a) an isolatedouabain-resistant Na⁺-K⁺-ATPase and/or reconstituted liposomescontaining or cells containing or expressing an ouabain-resistantNa⁺-K⁺-ATPase; (b) HIF; (c) a non-ATP substrate, such as NADH; (d) ATPand/or labeled ATP; and (e) a compound containing isotopic Rb⁺.

The following are examples of specific embodiments of the inventiondescribed herein. They are intended to serve as illustrations and notlimitations of the present invention.

EXEMPLIFICATION Example 1 Coupled-enzyme Assay

Na⁺-K⁺-ATPase activity is measured spectrophotometrically at about 37°C. as a decrease in absorbance at 340 nm due to of the oxidation ofNABH, as previously described (Haupert et al., Am. J. Physiol.247:F919-F924, 1984).

The test substance and HIF, typically in amounts of nanograms ormicrograms, are each individually preincubated with 0.5 μg of purifiedNa⁺-K⁺-ATPase at about 37° C. for about 30 minutes in a final volume of50 μl of preincubation mixture containing 100 mM NaCl, 3 mM MgCl₂, 50 mMtris(hydroxymethyl)aminomethane (Tris).HCl, pH 7.4. The inhibitoryactivity of the test substance and HIF is measured in the presence,during preincubation, of the following chelators: 2 mM norepinephrine, 2mg/ml BSA, and 1 mM ethylene glycol-bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) to avoid interferences byvanadate, liberated fatty acids, and bivalent cations (Carilli et al.,J. Biol. Chem. 260:1027-1031, 1985). After preincubation, 40 μl of theincubating solution are added to 1 ml of the prewarmed reading solution:100 mM NaCl, 25 mM KCl, 6 mM MgCl₂, 1.4 mM phosphoenolpyruvate, 1 mMdithiothreitol, 20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonicacid (HEPES), 3 mM ATP, 0.26 mM NADH, 10 μg/ml lactate dehydrogenase,and the absorbance is recorded at about 37° C. for at least 2 minutes.The inhibitory activity of the test substance and HIF is expressed aspercent of the control sample, carried out in the same conditions but inthe absence of the test substance and HIF.

Example 2 ATP Hydrolysis Assay

Na⁺-K⁺-ATPase activity is assayed detecting or measuring the release of³²P from [³²P]ATP, as previously described (Doucet et al., Am. J.Physiol. 237:F105-F113, 1979). Test substance and HIF, typically inamounts of nanograms or micrograms, are each individually preincubatedwith 0.3 μg of purified enzyme for 45 minutes at about 37° C. in 50 μlfinal volume of preincubation medium containing 140 mM NaCl, 3 mM MgCl₂,50 mM HEPES-Tis, pH 7.4. Parallel experiments are carried out, whenspecified, in the presence of different buffer solutions or chelatorsduring preincubation, such as 2 mM norepinephrine, 1 mM EDTA, 1 mM EGTA,1 mM glutathione, 5 mM mercaptoethanol and 2 mg/ml BSA, to evaluate theionic requirements for the binding of the test substance, and to findout if, in comparison to HIF, chelators interfere with the inhibitoryactivity of the test substance. After the preincubation period, 10 μl ofincubation solution, containing 10 mM KCl and 20 nCi [³²P]ATP (0.5-3Ci/mmol) are added, and the reaction is continued for about 15 minutesat about 37° C. The reaction is stopped by acidification with ice-coldperchloric acid solution at 30% vol/vol. ³²P is separated bycentrifugation with activated charcoal, and radioactivity is measured byliquid scintillation counting (Beckman LS 5000 CE). The inhibitoryactivity of the test substance and HIF is expressed as percent of thecontrol sample, carried out in the absence of the test substance andHIF.

Example 3 Ion Transport Assay

Purified Na⁺-K⁺-ATPase is reconstituted into phosphatidylcholineliposomes using the cholate-dialysis method disclosed by Anner andMoosmayer in Biophys. Res. Commun. 129:102-108, 1985. Cells containingan ouabain-resistant Na⁺-K⁺-ATPase, such as rat kidney cells or ratblood cells (erythrocytes) are prepared by the standard techniques knownin the art (Shyjan and Levenson, Biochemistry 28:4531-4535, 1989;Carilli et al., J. Biol. Chem., 260:1027-1031, 1985).

Ion transport activity of the reconstituted liposomes containing anouabain-resistant Na⁺-K⁺-ATPase, isolated kidney cells, or erythrocytes,which contain α1/β1 complex as the sole Na⁺-K⁺-ATPase, is assayed bypreviously described methods (Anner, et al., Am. J. Physiol.,258:F144-F153, 1990; Cantiello, et al., Am. J. Physiol., 255:F574-F580,1988; Crabos, et al., Am. J. Physiol., 254:F912-F917, 1988,respectively). Freshly reconstituted liposomes, isolated kidney cells orerythrocytes are washed with a physiological solution containing 140 mMNaCl, 1 mM CaCl₂, 1 mM MgSO₄, 20 mM HEPES, 5 mM Na₂HPO₄ and 10 mMglucose, pH 7.5, and resuspended (Anner et al., Am. J. Physiol.258:F144-F153, 1990).

The test substance and HIF, typically in amounts of nanograms ormicrograms, are each individually preincubated from about 30 minutes toabout 3 hours at about 0° C. (liposomes), about 37° C. (kidney cells orerythrocytes) with washed reconstituted liposomes, kidney cells orerythrocytes in a final volume of 2.5 μl (liposomes) 50 μl (kidneycells, erythrocytes, 25% final hematocrit in the case of erythrocytes).The preincubation is carried out both in the presence and absence of 5mM KCl. After this period, 0.3 μCi ⁸⁶RbCl (1.5 μCi/ml) and 5 mM KCl(when not present in the preincubation) are added. The incubation isstopped after 30 minutes by adding 100 to 500 μl ice-cold physiologicalbuffer solution. Parallel experiments are conducted in the absence ofeither the rest substance or HIF, but in the presence of 10⁻² M ouabainto allow determination of ion (Rb⁺) transport activity nor related tothe ouabain-resistant Na⁺-K⁺-ATPase. The liposomes are separated bychromatography. The kidney cells or erythrocytes are separated from themedium by rapid centrifugation through a 500 μl phthalate oil layer,after which the tips of the tubes are sliced off and counted for ⁸⁶Rbcontent (Beckman gamma counter 5500 B). The ouabain-sensitive ⁸⁶Rbuptake is calculated as the difference between the Rb uptake in theabsence and presence of 10⁻² M ouabain.

EQUIVALENTS

While this invention has been particularly shown and described withreference to certain embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the spirit and scope of the invention asdefined by the appended claims.

1. A method for screening a test substance other than ouabain, forHIF-like inhibitory activity of an ouabain-resistant Na⁺-K⁺-ATPase,comprising the steps of: (a) contacting the ouabain-resistantNa⁺-K⁺-ATPase with the test substance under suitable conditions formeasuring or detecting ouabain-resistant Na⁺-K⁺-ATPase activity; (b)measuring or detecting ATPase activity of the test substance; and (c)comparing the activity of the ouabain-resistant Na⁺-K⁺-ATPase measuredor detected in step (b) with the ouabain-resistant Na⁺-K⁺-ATPaseactivity of HIF under comparable conditions, thereby determining whetherthe test substance exhibits HIF-like inhibitory activity.
 2. The methodof claim 1, further comprising the steps of contacting ouabain-resistantNa⁺-K⁺-ATPase with HIF under suitable conditions in an aqueous mediumfor ouabain-resistant Na⁺-K⁺-ATPase activity and measuring or detectingthe activity, thereby providing the measurement or detection to becompared to the measurement or detection of the test substance.
 3. Themethod of claim 1 wherein the ouabain-resistant Na⁺-K⁺-ATPase is onewhich is isolated from a target cell.
 4. The method of claim 3, whereinthe target cell is selected from the group consisting of a kidney cell,a heart cell, a pineal gland cell, a skeletal muscle cell, a retinahorizontal cell, a retina Muller cell, a brain cortical astrocyte, acerebellar granule neuron, a cortical neuron and a Hippocampal neuron.5. A method for screening a test substance for HIF-like inhibitoryactivity of an ouabain-resistant Na⁺-K⁺-ATPase, comprising the steps of:(a) contacting the ouabain-resistant Na⁺-K⁺-ATPase with the testsubstance in the presence of ATP and a non-ATP substrate, under suitableconditions for measuring or detecting oxidation of the non-ATPsubstrate; (b) measuring or detecting oxidation of the non-ATPsubstrate; and (c) comparing the measured or detected oxidation of thenon-ATP substrate with oxidation of the non-ATP substrate in thepresence of HIF and ATP under comparable conditions, thereby determiningwhether the test substance exhibits HIF-like inhibitory activity.
 6. Themethod of claim 5, further comprising the steps of contactingouabain-resistant Na⁺-K⁺-ATPase activity with HIF under suitableconditions in an aqueous medium for ouabain-resistant Na⁺-K⁺-ATPaseactivity and measuring or detecting the activity, thereby providing themeasurement or detection to be compared to the measurement or detectionof the test substrate.
 7. The method of claim 5, wherein the oxidationlevel of the non-ATP substrate is measured by a decrease of the amountof the non-ATP substrate.
 8. The method of claim 5, wherein the non-ATPsubstrate is NADH.
 9. The method of claim 8, wherein the oxidation ofNADH is measured by a decrease of the amount of NADH.
 10. The method ofclaim 9, wherein the decrease of the amount of NADH is measured by adecrease in absorbance at 340 nm.
 11. The method of claim 1 or 5,wherein the HIF is isolated from bovine hypothalamus.
 12. The method ofclaim 1 or 5, wherein the ouabain-resistant Na⁺-K⁺-ATPase is selectedfrom the group consisting of a rodent, toad and butterfly α1Na⁺-K⁺-ATPase.
 13. The method of claim 12, wherein the ouabain-resistantNa⁺-K⁺-ATPase is a rat kidney α1 ouabain-resistant Na⁺-K⁺-ATPase. 14.The method of claim 12 wherein the ouabain-resistant Na⁺-K⁺-ATPase is aBufo marinus α1 ouabain-resistant Na⁺-K⁺-ATPase.
 15. The method of claim12 wherein the ouabain-resistant Na⁺-K⁺-ATPase is a Danus plexippus α1Na⁺-K⁺-ATPase.
 16. A method for screening a test substance other thanouabain, for HIF-like inhibitory activity of an ouabain-resistantNa⁺-K⁺-ATPase, comprising the steps of: (a) contacting theouabain-resistant Na⁺-K⁺-ATPase with the test substance in the presenceof ATP, wherein the terminal phosphate (P₃) is labeled, under suitableconditions for measuring or detecting liberated labeled P₃; (b)measuring or detecting liberated labeled P₃; and (c) comparing themeasured or detected liberated labeled P₃ with the measured or detectedlabeled P₃ that is liberated by contacting ouabain-resistantNa⁺-K⁺-ATPase with HIF in the presence of ATP, wherein the terminalphosphate is labeled, under comparable conditions, thereby determiningwhether the test substance exhibits HIF-like inhibitory activity. 17.The method of claim 16, further comprising the step of contactingouabain-resistant Na⁺-K⁺-ATPase with HIF under suitable conditions in anaqueous medium for ouabain-resistant Na⁺-K⁺-ATPase activity, andmeasuring or detecting the activity, thereby providing the measurementor detection to be compared to the measurement or detection of the testsubstance.
 18. The method of claim 16, wherein the label is selectedfrom the group consisting of a radiolabel, a fluorescent label, aluminescent label and an enzymatic label.
 19. The method of claim 16,wherein the ATP is [³²P]ATP.
 20. The method of claim 19, wherein theamount of liberated labeled ³²P is measured by liquid scintillationcounting.
 21. The method of claim 16, wherein the liberated, labeled P₃is separated from the reaction mixture.
 22. The method of claim 21,wherein the separation is by centrifugation.
 23. The method of claim 22,wherein the centrifugation is carried out in the presence of activatedcharcoal.
 24. The method of claim 16, wherein the ouabain-resistantNa⁺-K⁺-ATPase is selected from the group consisting of a rodent, toadand butterfly α1 Na⁺-K⁺-ATPase.
 25. The method of claim 24, wherein theouabain-resistant Na⁺-K⁺-ATPase is a rat kidney α1 ouabain-resistantNa⁺-K⁺-ATPase.
 26. The method of claim 24 wherein the ouabain-resistantNa⁺-K⁺-ATPase is a Bufo marinus α1 ouabain-resistant Na⁺-K⁺-ATPase. 27.The method of claim 24 wherein the ouabain-resistant Na⁺-K⁺-ATPase is aDanus plexippus α1 Na⁺-K⁺-ATPase.
 28. The method of claim 16 wherein theouabain-resistant Na⁺-K⁺-ATPase is one which is isolated from a targetcell.
 29. The method of claim 28 wherein the target cell is selectedfrom the group consisting of a kidney cell, a heart cell, a pineal glandcell, a skeletal muscle cell, a retina horizontal cell, a retina Mullercell, a brain cortical astrocyte, a cerebellar granule neuron, acortical neuron and a Hippocampal neuron.
 30. A kit comprising: (a) anisolated ouabain-resistant Na⁺-K⁺-ATPase; (b) ATP; (c) NADH; and (d)HIF.